Impact of Sporisorium scitamineum infection on the qualitative traits of commercial cultivars and advanced lines of sugarcane

Whip smut disease of sugarcane, caused by Sporisorium scitamineum, is considered one of the main constraints in the successful cultivation of sugarcane. The pathogen infection can decrease the quantity and quality of the produce. Cultivation of resistant varieties is the most feasible strategy to combat the harms of this devastating disease. Development of varieties having disease-resistance together with improved important traits such as brix, pol, purity, CSS, and low fiber contents are desirable. Therefore, we documented the variances in quality traits of 104 sugarcane cultivars under disease pressure in split-plot design with 6 replications. The split ANOVA revealed a highly significant impact (p<0.0001) between treatments (inoculated and uninoculated), within cultivars as well as interaction ‘Cultivars x Treatments’ effect on brix, pol, fiber, purity, and CSS contents. In inoculated plots, the infection of S. scitamineum brought a highly significant reduction (t>4.032) in brix, pol, purity, and CSS of more than 40% of the cultivars used, as compared to the uninoculated ones. On the other hand, the smut infection caused a highly significant (t>4.032) increase in fiber percentage of 41 cultivars. We found significant positive correlations between smut rating and reduction of brix, pol, purity, and CSS contents. The cultivars that were caught with greater disease severity, compromised a higher reduction of their useful contents. Similarly, a significant positive correlation was found between increased fiber percent and smut rating. Remarkably, cultivars that showed immune reactions to whip smut disease were not statistically different from uninoculated ones in brix, pol, purity, CSS, and fiber contents. Variable effects of whip smut infection to quality parameters of different cultivars depict the importance of further improvement through breeding programs.


Introduction
Sugarcane (Saccharum officinarum L.) is classified in the grass family Poaceae along with other economically important plant species including wheat, rice, barley, oats, rye, etc. It is the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 experimental site has a long history of wheat-cotton rotation, which means no cane cultivation in past. This study was undertaken for two consecutive seasons (2014-15 and 2015-16).
Indigenous and exotic sugarcane germplasm were collected from different sources such as National Sugar & Tropical Horticulture Research Institute (NSTHRI) Thatta, Nuclear Institute of Agriculture (NIA) Tandojam and Agriculture Research Institute (ARI) Tandojam (Table 1). Planting material was obtained from a one-year old crop, which was completely free from smut disease. To minimize the chances of contamination of planting materials with soil-borne propagules of smut pathogen, before treatment and sowing all planting materials were subjected to hot water treatment (52˚C for 30 minutes) to eliminate any setts borne pathogen inoculum [34].

Land preparation and experimental design
S. scitamineum is a soil-borne pathogen, therefore, the experiment was carried out in the soil which had no previous history of sugarcane plantation and arranged in a split-plot design. This was done to consider that the soil was free from S. scitamineum and to avoid cross-contamination. The soil texture and chemical properties are best suited for cane cultivation. Prior to sowing, all standard agronomic practices were followed to prepare the land. A total of 104 cultivars of different origins were grown with 6 replications in which disease treatments (inoculated & uninoculated) served as whole plots (Factor A) and cultivars as sub-plots (Factor B), randomized within each block. Each subplot consisted of a single row of 5-meter length, with a row to row distance of one meter. About 13 (3-budded) setts of each cultivar were sown in the ridges so that the standard seed rate of 80000 buds per hectare was achieved [35,36]. The length of each block was measured from the first row to the last row of the plot, which made the plot size 5×104×6 = 3120 square meters excluding paths, etc.

Pathogen inoculation
Smut whips were collected from infected sugarcane fields of all the cane growing regions of the Sindh province to ensure the presence of all S. scitamineum present in a study area. The teliospores were gently scraped from shade-dried whips and stored in the refrigerator for future use. At the time of inoculation, their viability was confirmed on water agar plates to be 90% [37]. For inoculation, spore suspension was prepared in large sterilized stainless steel containers, filled with 50 liters of distilled sterilized water, amended with teliospores, and a few drops of Tween 20 to homogenize the suspension. The inoculum density was set to 5×10 6 spores ml -1 with the help of a hemocytometer [38]. The three budded setts of each cultivar were dipped into spore suspension for 30 minutes and then kept in polythene bags for overnight to provide favorable conditions for spore penetration [17,39,40]. The sets dipped in distilled-sterilized water having no pathogen inoculum, served as control (un-inoculated).

Qualitative observation
The trial was harvested 14 months after planting, at the time when sucrose accumulation in the cultivars was optimal. The qualitative parameters such as brix, pol, purity, fiber, and CCS were calculated by taking five stalks per furrow. The canes were crushed with the help of a Cutter grinder (Fabricator) (Model No. SCF-L4, Smith Crafts Fabricator, Gujranwala, Pakistan) to obtain at least 2 kg of crushed material for quality analysis. Five hundred grams of crushed canes were pressed in the hydraulic press (Model No. SCF-HP-06, Smith Crafts Fabricator, Gujranwala, Pakistan); the yielded sugar juice was collected in a 500 ml glass beaker and fiber cake was removed to calculate fiber contents.
Fiber. The press cape residues were weighed to find out the bagasse percent of cane and a sub-sample of the same material was used to calculate the moisture percentage in bagasse. 100 grams of the material from the press cape residue was utilized and placed in a Petri dish. The Petri dish plate was then deposited in an oven and dried at a temperature of 110 to 120 0 F for about 90 to 120 minutes until the bagasse weight become constant. The loss in bagasse weight after drying provides the percentage of moisture in bagasse and fiber percent was determined as suggested by Chen and Chou [42]: Brix. A cane juice sample was collected in a 500 ml beaker. A drop of juice was placed on the prism of the Refractometer (PR-101, ATAGO Co. Ltd, Japan) with the help of a pipette to measure the brix percentage of the sample.
Purity. The purity of cane juice is measured for an idea about the maturity of the cane sample or deterioration by the following equation [43]: Commercial Cane Sugar (CCS). CCS of the samples determined by using the following formula [44]: Where P stands for the pol percentage of the first-expressed juice, B is the brix percentage of the first-expressed juice and F is the fiber percentage in the cane.

Data analysis
Statistical parameters such as mean, standard deviation, analysis of variance, LSD multiple comparison tests, paired t-test, and regression equations were calculated by using the Statistix-8.1 analytical software.

Effects of S. scitamineum on brix
Analysis of variance showed that for brix, there was a highly significant difference between treatments (DF = 1, F = 443.98, P = 0.0000), among cultivars (DF = 103, F = 108.07, P = 0.0000) and the interactive response of treatments and cultivars (DF = 103, F = 11.22, P = 0.0000) ( Table 2). In aggregate, significantly higher mean brix was recorded in un-inoculated plots (21.81%) as compared to the pathogen-inoculated treatments (20.69%) ( Table 3). Brix % of all 104 cultivars in-field screening trial in both treatments, as well as calculated reduction and T-values are given in S1 Table. In inoculated plots, maximum mean brix was recorded in sugarcane cultivars BPTh-807 (23.31±0.08%) followed by HoTh-318 (23.23 There was no significant difference between brix percent of inoculated and un-inoculated cultivars, which were immune (S1 Table).
The least-square linear regression analysis (Fig 1) showed that the smut rating and the reduction percentage of brix were moderately related to each other (R 2 = 0.6662) and disease rating is influencing 67% reduction of brix of sugar cane. The statistical analysis further indicated that there is a significant (p<.001) effect of disease incidence on brix. Moreover, according to the regression equation (y = 1.3728x + 0.3583), it can be predicted that with the increase of single rating in the disease, 1.3728 percent of variation can be observed in brix percentage.
In the case of the effect of smut rating and the reduction in the percentage of pol (Fig 2), the graph revealed that there was a positive and highly significant relationship (R 2 = 0.7703, p<.001) between the disease and pol. The regression equation further showed that with an increase of one rating in disease may vary pol up to 1.8923%.

Effects of whip smut S. scitamineum on fiber contents
On an overall basis, cultivars showed significant impact (DF = 103, F = 84.51, P = 0.000). Moreover, pathogen infection also adversely affected the fiber contents in susceptible cultivars.  The pathogen treatment's effect was also highly significant (DF = 1, F = 446.87, P = 0.000). The interactive effect of cultivars and treatments also appeared highly significant (DF = 103, F = 11.37, P = 0.000) ( Tables 3 and 5 .23% more fiber as compared to uninoculated. In comparison to un-inoculated plants, a maximum increase in fiber contents was found in inoculated plants of cv. HoTh-550 (13.23%), HoTh-408 (12.72%), CP-29-120 (12.08%) and Tritan (11.81%). About 21 cultivars showed moderate reaction to smut pathogen and produced 2.86 to 5.06% increased fiber content as compared to uninoculated. However, there was no significant (t<2.571) difference in fiber recorded in 42 cultivars among inoculated and un-inoculated planting materials (S3 Table).  The regression analysis equation (y = 1.1668 x + 0.27665) in (Fig 3) for predicting the influence of smut disease rating on fiber content, showed a positive and significant (p<.001) relationship between the disease rating and fiber. The trend line and the value of R 2 (0.6220) revealed the goodness of the regression model.
The results regarding the relationship of the reduction percentage of CCS and intensity of disease through regression analysis (Fig 5) exhibited that the CCS of sugarcane cultivars was greatly influenced by the rating of smut disease. There was a strong (R 2 = 0.7924) and a highly significant (p<.001) relationship between the two variables. The calculated regression equation (y = 2.0541x -0.4976) forecast that each unit increase in smut rating causes a 2.0541 percent reduction in the CCS of sugarcane.

Discussions
Sugarcane smut is widespread in almost all cane-growing areas of the world and considered one of the most important factors in varietal development programs. A high level of  susceptibility to smut has forced to stop the commercial cultivation of many high-yielding varieties [45][46][47]. The infection of S. scitamineum not only reduced physical cane yield but also brought a significant reduction in the quality of cane juice, the ultimate product [24,48]. However, the extent of losses depends upon disease severity, which is influenced by climatic factors and cultivars [21,22,49]. Searching of cultivars, which performed better in terms of all desirable parameters in the presence of smut pathogen, is an ongoing process. Besides quantitative traits, the impact of different diseases on juice quality parameters has been the main concern of scientists [50][51][52][53]. In the present investigation in which effects of S. scitamineum and different cultivars on qualitative parameters were assessed, it appears that pathogen treatment's main effect, as well as cultivars main effects caused a highly significant impact on all tested quality characteristics. In aggregate, brix, pol, purity, and CSS contents significantly decreased in smut inoculated plots, while fiber tends to enhance. The regression analysis also indicates a strong and positive relationship between increased disease rating and quality parameters. In inoculated plots, cultivars produced an immune or resistant response to smut pathogen, showed no or less adverse impact on quality traits. On the other hand, in highly susceptible cultivars maximum reduction of 16.34% in brix, 25% in pol, 10.42% in purity, and 32% in CSS. While fiber contents in susceptible cultivars increased up to 13%. Many other workers also found a significant reduction in cane juice quality along with quantitative losses due to smut disease [31,54,55]. However, the extent of losses mainly depends upon the varieties under cultivation. Alexander [56] and Thurston [57] calculated 15-20% yield and quality losses under moderate levels of disease. More specifically, 32% losses in quality [26] and 22.2% in sucrose content were also noted in smut infected canes [58]. Our findings are in accordance with Kumar et al. [32] and Irvine [30] who also observed a significant reduction in sucrose; purity, brix, and viscosity due to smut infection. Besides whip smut, other biotic stresses such as red rot and wilt diseases also caused adverse effects on qualitative traits. In susceptible cane cultivars, wilt disease caused a reduction of 44.48% in brix, 59% in pol, 25.7% in purity, and 66% in CSS [52]. Similarly, varietal screening and use of control measures against red rot disease have been continuously carried out due to its negative impact on qualitative parameters [59][60][61]. In India, red rot disease caused 20.04-38.79% reduction in brix, (17.68-31.56% in purity 29.06-53.57% in CSS [51]. The findings of the present studies confirmed that sugarcane juice quality characters were greatly affected by the level of smut infection in a particular cultivar. Moreover, comprehensive field testing of cultivars objectively revealed the demonstrated the cane cultivars performed well in all qualitative parameters in the presence of smut pathogen.
Supporting information S1